Efficient refolding of proteins in vitro is an important problem in protein structural analysis and biotechnological manufacturing of pharmaceutical products. Because of their inherent ability to rapidly overexpress proteins to high yields, bacterial systems are the organisms of choice for protein mass production. Unfortunately, overexpression of foreign and, especially, mutant proteins often leads to the development of large intracellular aggregates or inclusion bodies (Rudolph, R and Lilie, H. (1996) FASEB J. 10, 49-56; Guise, A. D., West, S. M., and Chaudhuri, J. B. (1996) Mol. Biotechnol. 6, 53-64, the disclosures of which are incorporated herein by reference). In some cases, the proper intracellular folding of the overexpressed proteins can be enhanced by lowering the cell growth temperature, co-expressing molecular chaperones, or introducing low molecular weight additives (Kujau, M. J., Hoischen, C., Riesenberg, D., and Gumpert, J. (1998) Appl. Microbiol. Biotechnol. 49, 51-58; Tate, C. G., Whiteley, E., and Betenbaugh, M. J. (1999) J. Biol-Chem. 274, 17551-17558; Minning, S., Schmidt-Dannert, C., Schmid, R. D. (1998) J. Biotechnol. 66, 147-156, the disclosures of which are incorporated herein by reference). More often, however, investigators are forced to rely on in vitro folding methods to denature (also known as “deactivate”) and then refold (also known as “reactivate”) aggregated proteins. A number of in vitro approaches have been developed to minimize protein aggregation and enhance proper refolding. Among those are: (1) the addition of osmolytes and denaturants to refolding buffer (Tate, C. G., Whiteley, E., and Betenbaugh, M. J. (1999) J. Biol-Chem. 274, 17551-17558; Plaza-del-Pino, I. M. and Sanchez-Ruiz, J. M. (1995) Biochemistry 34, 8621-8630, Frye, K. J. and Royer, C. A. (1997) Protein. Sci. 6: 789-793, the disclosures of which are incorporated herein by reference); (2) the use of the combinations of different molecular chaperones (Thomas, J. G., Ayling, A., and Baneyx, F. (1997) Appl. Biochem. Biotechnol. 66, 197-238; Buchberger, A., Schroder, H., Hesterkamp, T., Schonfeld, H. J., and Bukau, B. (1996) J. Mol. Biol. 261, 328-233; Veinger, L., Diamant, S., Buchner, J., and Goloubinoff, P. (1998) J. Biol. Chem. 273, 11032-11037, the disclosures of which are incorporated herein by reference); (3) immobilization of folding proteins to matrices and matrix-bound chaperonins (Stempfer, G., Holl-Neugebauer, B., and Rudolph, R. (1996) Nat. Biotechnol. 14, 329-334; Altamirano, M. M., Golbik, R., Zahn, R., Buckle, A. M., and Fersht, A. R. (1997) Proc. Natl. Acad. Sci. USA 94, 3576-3578; Preston, N. S., Baker, D. J., Bottomley, S. P., and Gore, M. G. (1999) Biochim. Biophys. Acta 1426, 99-109, the disclosures of which are incorporated herein by reference); and (4) utilization of folding catalysts such as protein disulfide isomerase and peptidyl-prolyl cis-trans isomerase (Altamirano, M. M., Garcia, C., Possani, L. D., and Fersht, A. R. (1999) Nat. Biotechnol. 17, 187-191, the disclosure of which is incorporated herein by reference). Unfortunately, because of the diversity of the protein folding mechanisms, there is no universal procedure for protein folding and folding conditions have to be optimized for each specific protein of interest. Therefore, there is always a need for new and more versatile folding techniques. This invention involves a novel protein folding procedure that combines the use of the GroE chaperonins and cellular osmolytes.
Because of its ability to bind many different protein folding intermediates, it was thought that the bacterial GroE chaperonin system could provide a general method to refold misfolded proteins. Chaperonin GroEL is a tetradecamer of identical 57 kDa subunits that possesses two large hydrophobic sites capable of binding to transient hydrophobic protein folding intermediates. The hydrophobic binding site undergoes the multiple cycles of exposure and burial driven by the ATP binding and hydrolysis and the co-chaperonin GroES binding and dissociation. Accordingly, the protein folding intermediates can undergo multiple rounds of binding to and release from the GroEL until they achieve the correctly folded state (for review, see Fenton, W. A. and Horwich, A. L. (1997) Protein Sci. 6, 743-760, the disclosure of which is incorporated herein by reference). Besides simple prevention of non-productive aggregation, chaperonins may also influence the conformation of the folding intermediates, actively diverting them to a productive folding pathway (Fedorov, A. N. and Baldwin, T. O. (1997) J. Mol. Biol. 268, 712-723; Shtilerman, M., Lorimer, G., and Englander, S. W. (1999) Science 284, 822-825, the disclosures of which are incorporated herein by reference). However, despite the general nature of chaperonin-protein interactions, there are many proteins that, for reasons that are currently unknown, cannot fold correctly from the bacterial chaperonin system.
The addition of osmolytes often results in an observed increase in stability of the native structure for some proteins. The stabilization effect is observed with various osmolytes and small electrolytes such as sucrose, glycerol, trimethylamine N-oxide (TMAO), potassium glutamate, arginine and betaine (Wang, A. and Bolen, D. W. (1997) Biochemistry 36, 9101-9108; De-Sanctis, G., Maranesi, A., Ferri, T., Poscia, A., Ascoli, F., and Santucci, R. (1996) J. Protein. Chem. 15, 599-606; Chen, B. L. and Arakawa, T. (1996) J. Pharm. Sci. 85, 419-426; Zhi, W., Landry, S. J., Gierasch, L. M., and Srere, P. A. (1992) Protein Science 1, 552-529, the disclosures of which are incorporated herein by reference). This effect is based on the exclusion of osmolytes from hydration shells and crevices on protein surface (Timasheff, S. N. (1992) Biochemistry 31, 9857-9864, the disclosure of which is incorporated herein by reference) or decreased solvation (Parsegian, V. A., Rand, R. P., and Rau. D. (1995). Methods. Enzymol. 259, 43-94, the disclosure of which is incorporated herein by reference). In a series of quantitative studies, Wang and Bolen have shown that the osmolyte-induced increase in protein stability is due to a preferential burial of the polypeptide backbone rather than the amino acid side chains (Wang, A. and Bolen, D. W. (1997) Biochemistry 36, 9101-9108). Because native protein conformations are stabilized, proper folding reactions are also enhanced in the presence of osmolytes (Frye, K. J. and Royer, C. A. (1997) Protein. Sci. 6: 789-793; Kumar, T. K., Samuel, D., Jayaraman, G., Srimathi, T., and Yu, C. (1998) Biochem. Mol. Biol. Int. 46, 509-517; Baskakov, I. and Bolen, D. W. (1998) J. Biol. Chem. 273: 4831-4834, the disclosures of which are incorporated herein by reference). Osmolytes usually affect protein stability and folding at physiological concentration range of 1-4 M (Yancey. P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., and Somero, G. N. (1982) Science 217, 1214-1222, the disclosure of which is incorporated herein by reference). However, it is apparent that the degree of stabilization depends on both the nature of the osmolyte and the protein substrate (Sola-Penna, M., Ferreira-Pereira, A., Lemos, A. P., and Meyer-Fernandes, J. R. (1997) Eur. J. Biochem. 248, 24-29, the disclosure of which is incorporated herein by reference) and, in some instances, the initial aggregation reaction can actually accelerate in the presence of osmolytes (Voziyan, P. A. and Fisher M. T. (2000) Protein Science, Volume 9, 2405-2412).
Although GroE chaperonins and osmolytes have been used in the folding protocols separately, no studies have taught or suggested the feasibility of combining these two approaches. This invention demonstrates that the combination of chaperonins and osmolytes can provide a considerable advantage in assisting protein folding. Moreover, the method of the present invention can be applied as a more general technique for a rapid identification of the optimal folding solution conditions to achieve maximal yields of correctly folded protein. In particular, the initial off-pathway aggregation is avoided through formation of stable chaperonin-protein substrate complexes under the solution conditions that favor the maximum binding of the substrate to GroEL. These long-lived stable complexes are added to a series of different osmolyte solutions (“folding array”) to identify the most efficient folding conditions for the protein substrate in question.
As a model, this invention examines the in vitro refolding of C-terminal truncation mutant of bacterial glutamine synthetase, GSΔ468. Unlike native glutamine synthetase (“GS”), this single amino acid truncation product folds to an intermediate that cannot be refolded to an active form by either chaperoning or osmolytes alone. However, the combination of chaperonins and a number of natural osmolytes allowed for the refolding of GSΔ468. Under the optimized conditions, close to 70% of mutant protein refolded to an active form, even at protein concentrations approaching 1 mg/ml.
Therefore, it is an object of this invention to provide an in vitro protein folding process for preventing large-scale protein misfolding and aggregation.
It is a further object to provide a protein folding process that concentrates aggregation prone chaperonin-protein folding intermediates in a stable non-aggregating form.
It is another object of this invention to provide a protein folding process that rapidly screens stable chaperonin-substrate intermediates for the best folding solution conditions.
To accomplish the above and related objects, this invention may be embodied in the detailed description that follows, together with the appended drawings and claims.